Eap1p, a novel eukaryotic translation initiation factor 4E-associated protein in Saccharomyces cerevisiae

Abstract

Ribosome binding to eukaryotic mRNA is a multistep process which is mediated by the cap structure [m(7)G(5')ppp(5')N, where N is any nucleotide] present at the 5' termini of all cellular (with the exception of organellar) mRNAs. The heterotrimeric complex, eukaryotic initiation factor 4F (eIF4F), interacts directly with the cap structure via the eIF4E subunit and functions to assemble a ribosomal initiation complex on the mRNA. In mammalian cells, eIF4E activity is regulated in part by three related translational repressors (4E-BPs), which bind to eIF4E directly and preclude the assembly of eIF4F. No structural counterpart to 4E-BPs exists in the budding yeast, Saccharomyces cerevisiae. However, a functional homolog (named p20) has been described which blocks cap-dependent translation by a mechanism analogous to that of 4E-BPs. We report here on the characterization of a novel yeast eIF4E-associated protein (Eap1p) which can also regulate translation through binding to eIF4E. Eap1p shares limited homology to p20 in a region which contains the canonical eIF4E-binding motif. Deletion of this domain or point mutation abolishes the interaction of Eap1p with eIF4E. Eap1p competes with eIF4G (the large subunit of the cap-binding complex, eIF4F) and p20 for binding to eIF4E in vivo and inhibits cap-dependent translation in vitro. Targeted disruption of the EAP1 gene results in a temperature-sensitive phenotype and also confers partial resistance to growth inhibition by rapamycin. These data indicate that Eap1p plays a role in cell growth and implicates this protein in the TOR signaling cascade of S. cerevisiae.

FIG. 1

Interaction of S. cerevisiae proteins with eIF4E. Yeast strains were grown to exponential phase and lysed by the glass bead method. Total protein (30 μg) from the clarified extracts was fractionated by SDS-PAGE and transferred to nitrocellulose membranes, which were then probed with ³²P-labeled HMK-eIF4E. (A) SDS-PAGE (8% gel). Strains used: lane 1, YCG323 (wild type [wt]); 2, YCG324 (tif4631::LEU2); 3, YCG325 (tif4632::URA3). (B) SDS-PAGE (15% gel). Lane 1, YMA-4B (wild type); 2, YMA-2A (caf20::URA3). The deduced identity of each of the eIF4E-interacting proteins is indicated with an arrow on the right. Positions of molecular mass standards (in kilodaltons) are marked on the left.

FIG. 2

(A) Predicted amino acid sequence of Eap1p in single-letter code. The p20 homology region is boxed, a potential bipartite nuclear localization sequence is in bold, a Walker A motif is underlined, and proline stretches in the C-terminal region are indicated by bullets above the sequence. (B) Limited sequence alignment between Eap1p and p20. Identical (black box) and conserved (shaded box) amino acids are highlighted. The alignment of critical residues common to mammalian 4E-BPs is shown below (x, any amino acid; Φ, hydrophobic residue).

FIG. 3

EAP1 encodes a novel 84-kDa eIF4E-interacting protein. Yeast extracts were prepared and analyzed by far-Western blotting as described for Fig. 1 except that samples were resolved by SDS-PAGE on a 10% gel. Strains used: lane 1, JK9-3da (wild type [wt]); 2, YGC034 (eap1::TRP1); 3, SH12-1A (caf20::URA3); 4, YGC047 (eap1::TRP1 caf20::URA3). The eIF4E-interacting proteins are indicated by arrows on the right. Positions of molecular mass standards (in kilodaltons) are marked on the left. DF, dye front.

FIG. 4

Mapping of the eIF4E interaction domain of Eap1p in vitro. Full-length (wild-type [wt]) EAP1 and deletion mutants of EAP1 were translated in vitro and incubated with either purified GST or GST-eIF4E prior to the addition of glutathione-Sepharose beads. Following extensive washing, the bound material was eluted by boiling in Laemmli buffer and resolved by SDS-PAGE (10% gel). (A) Schematic diagram of deletion mutants used in the study. The p20 homology region (aa 109 to 121) is indicated as a shaded box, the Walker A motif is shown as a stippled box, and the proline-rich region is cross-hatched. (B) Coprecipitation analysis. Load, one-fifth of total radiolabeled Eap1p used in the coprecipitation; GST, Eap1p coprecipitated by GST alone; GST-eIF4E, Eap1p coprecipitated by GST-eIF4E fusion protein. Full-length translation products are indicated by dots. Sizes of standards are indicated in kilodaltons.

FIG. 5

In vivo interaction of Eap1p with eIF4E is dependent on the 4E-binding motif. Yeast strain YGC034 (eap1::TRP1) was transformed with vector YEp352 alone or with the same vector expressing either HA-Eap1p or the HA-Eap1p[Y109A] mutant. Yeast were grown to exponential phase and lysed by the glass bead method. Protein (100 μg) was incubated with anti-HA monoclonal antibody before the addition of protein G-Sepharose beads. Following extensive washings, the bound proteins were resolved by SDS-PAGE, transferred to nitrocellulose membranes, and immunoblotted against anti-HA antibody (to detect HA-Eap1p; indicated by a dot) (A) or anti-yeast eIF4E monoclonal antibody 9B12 (B). Load, an equal amount of extract used for the coimmunoprecipitation loaded directly onto the gel; Free, proteins remaining in supernatant following immunoprecipitation; Bound, proteins adsorbed to the resin. IgG, immunoglobulin G heavy chain. Sizes of standards are indicated in kilodaltons.

FIG. 6

Eap1p competes with eIF4G and p20 for binding to eIF4E. Yeast extract was prepared as described for Fig. 5. Protein (100 μg) was incubated with m⁷GDP-agarose, and bound proteins were resolved by SDS-PAGE. (A) Western blotting was performed using anti-eIF4E monoclonal antibody 9B12. (B) Far-Western blotting was conducted using ³²P-labeled HMK-eIF4E as a probe. The eIF4E-interacting proteins are identified with arrows on the right. The asterisk indicates a degradation product of eIF4G2 which was observed sporadically in crude yeast extracts. Note that Eap1p[Y109A] mutant is not revealed by this analysis (see text). Free, proteins remaining in supernatant following immunoprecipitation; Bound, proteins adsorbed to the resin. Sizes of standards are indicated in kilodaltons.

FIG. 7

In vitro translation in yeast cell extract. (A) Recombinant Flag-tagged Eap1p was immunopurified from insect cells. The purified protein was resolved by SDS-PAGE and revealed by Coomassie staining or by far-Western analysis using ³²P-labeled eIF4E. Sizes of molecular weight (MW) markers are indicated in kilodaltons. (B) Translation reactions in an extract generated from YGC034 (eap1::TRP1) were conducted as described in Materials and Methods. Increasing amounts of recombinant Flag-Eap1p were added to the reaction mixtures, and translation was initiated with 100 ng of capped CAT mRNA containing or lacking the Ω sequence. Samples were fractionated by SDS-PAGE, and CAT protein was revealed by autoradiography. (C) Data shown in panel B were quantitated by densitometry and normalized to the amount of CAT synthesis in the absence of added Eap1p. The results are a representative of two independent experiments which did not vary significantly.

FIG. 8

Disruption of EAP1 confers a temperature-sensitive phenotype. Yeast strain YGC034 (eap1) was transformed with either YCplac33 (vector), pTS115 (EAP1), or pTS117 (EAP1[Y109A]). Resulting transformants and wild-type strain JH6-1C (wt) were streaked on YPD media and incubated at either 30 or 39°C for 2 and 3 days, respectively.

FIG. 9

Disruption of EAP1 confers partial resistance to rapamycin. (A) Yeast strains JH11-1C (TOR1-1), JH6-1C (wild type [wt]), and YGC034 (eap1) were streaked on YPD alone and on YPD containing rapamycin (20 ng/ml) and incubated at 30°C. (B) Indicated yeast strains were transformed with YCplac33 (vector), pTS115 (EAP1), or pTS117 (EAP1[Y109A]). Resulting transformants were streaked on SD-Ura medium and SD-Ura medium containing rapamycin (1 ng/ml) and then incubated at 30°C. tor1, strain AS93-2A; tor1 eap1, strain TS6-5A.